VEGF overexpressed mesoangioblasts enhance urethral and vaginal recovery following simulated vaginal birth in rats

Vaginal birth causes pelvic floor injury which may lead to urinary incontinence. Cell therapy has been proposed to assist in functional recovery. We aim to assess if intra-arterial injection of rat mesoangioblasts (MABs) and stable Vascular Endothelial Growth Factor (VEGF)-expressing MABs, improve recovery of urethral and vaginal function following simulated vaginal delivery (SVD). Female rats (n = 86) were assigned to either injection of saline (control), allogeneic-MABs (MABsallo), autologous-MABs (MABsauto) or allogeneic-MABs transduced to stably expressed VEGF (MABsallo-VEGF). One hour after SVD, 0.5 × 106 MABs or saline were injected into the aorta. Primary outcome was urethral (7d and 14d) and vaginal (14d) function; others were bioluminescent imaging for cell tracking (1, 3 and 7d), morphometry (7, 14 and 60d) and mRNAseq (3 and 7d). All MABs injected rats had external urethral sphincter and vaginal function recovery within 14d, as compared to only half of saline controls. Functional recovery was paralleled by improved muscle regeneration and microvascularization. Recovery rate was not different between MABsallo and MABsauto. MABsallo-VEGF accelerated functional recovery and increased GAP-43 expression at 7d. At 3d we detected major transcriptional changes in the urethra of both MABsallo and MABsallo-VEGF-injected animals, with upregulation of Rho/GTPase activity, epigenetic factors and dendrite development. MABSallo also upregulated transcripts that encode proteins involved in myogenesis and downregulated pro-inflammatory processes. MABsallo-VEGF also upregulated transcripts that encode proteins involved in neuron development and downregulated genes involved in hypoxia and oxidative stress. At 7d, urethras of MABsallo-VEGF-injected rats showed downregulation of oxidative and inflammatory response compared to MABSallo. Intra-arterial injection of MABsallo-VEGF enhances neuromuscular regeneration induced by untransduced MABs and accelerates the functional urethral and vaginal recovery after SVD.


Scientific Reports
| (2023) 13:8622 | https://doi.org/10.1038/s41598-023-35809-x www.nature.com/scientificreports/ Pelvic floor dysfunction (PFD) is a condition with significant physical, mental and social consequences for women worldwide. Childbirth and pregnancy are crucial contributing factors in its pathophysiology. Delivery is a serious traumatic event, causing anatomical and functional changes in the pelvic floor. Passage of the fetal head causes high and sustained pressure and deformation of the pelvic floor 1 leading to both ischemia and reperfusion and stretch-related injury to nerves, muscles and the supporting structures 2 . Whereas PFD may be present early after delivery, in some women these dysfunctions persist or resurface at a later age. Hypothesizing that symptomatic women may be those who incompletely recover morphologically from the trauma caused by vaginal delivery, a secondary prevention strategy by cell therapy administration around the time of the trauma has been suggested in patients with stress urinary incontinence or being at high risk for it 2 , such as previous incontinence, prolonged second stage of labour, high birth weight or any indication of severe trauma evidenced by ultrasound.
In rats, the effects of vaginal birth can be simulated by vaginal distension (VD) 3 and/or pudendal nerve crush (PNC) 4 . VD mimics the mechanical forces of vaginal delivery and PNC simulates the damage to the pudendal nerve occurring during childbirth. The combination of VD and PNC creates longer lasting functional and anatomical effects 5,6 , whereafter urethral function recovers by 21d postpartum evidenced by the frequency of external urethral sphincter (EUS) bursting 4 . Despite functional recovery, structural changes persist 9 weeks after SVD 5 . Beside urethral injury, SVD also induces structural and functional vaginal damage in rats 4 .
Several studies have been done to document the effects of different stem cell types on pelvic floor function recovery after vaginal birth [7][8][9][10] . Administration of stem cells after VD increased urinary LPP as well as the density of vessels, elastin and smooth muscle surrounding the urethra 8,11 . Mesoangioblasts (MABs) are vessel-derived stem cells with a high regenerative potential for muscular disorders 12,13 and could therefore be good candidates for repairing birth injury. Therefore, we aimed to investigate their effects in a standardized animal model for SVD. Based on our previous findings, we have selected intra-arterial delivery, which resulted in the highest amount of and more homogenous distribution of MABs in relevant pelvic organs 14 .
MABs release several growth factors, such as VEGF, an angiogenic protein with therapeutic potential in ischemic disorders 15 . Moreover, VEGF is also pro-myogenic 16,17 , neuroprotective 18 and induces nerve regeneration 19 . Therefore, it is reasonable to postulate that increased VEGF secretion by MABs may enhance the regeneration of the pelvic floor structures recovering from vaginal birth injury. The advantages of using genetically modified stem cells as vehicles include the fact that they can ensure a continuous and concentrated local supplementation of diffusible therapeutic molecules, reducing off-target delivery and also the side-effects of systemic administration 20 . MABs show a high adhesion-dependent migratory capacity and can reach perivascular targets especially in damaged areas 21 . Thus, MABs may be effective production sites to ensure stable VEGF delivery, promoting differentiation, survival, and functionality of neurons and muscle cells. Herein we also aimed to evaluate the potential of stable VEGF-expressing MABs in promoting nerve and muscle regeneration of urethra and vagina after SVD injury in rats.

Materials and methods
Study design. This study was set up to evaluate the effect of intra-arterial injected MABs in the recovery of the urethral sphincter and the vagina following SVD. The primary outcome was urethral function, evidenced by the frequency of bursting of the EUS during micturition, as measured by micro-ultrasound 22 . This study consists of two phases. First, we investigated how different types of MABs (allogeneic, autologous and VEGFoverexpressed allogeneic cells) boost urogenital functional recovery and investigate their fate in the pelvic region ( Fig. 1). Second, we selected relevant groups for further morphological and genetic characterization, to understand the mechanism of the observed functional improvement by VEGF overexpression observed. This study is reported in accordance with ARRIVE guidelines.
Mesoangioblasts. Skeletal muscles from both hind limbs were harvested, processed and characterized as previously described 14 . To enable tracking MABs after injection in vivo and in real time, and to prove cell viability, MABs were transduced with a lentiviral vector LV_CMV-eGFP-T2A-fLuc. For MABs allo-VEGF treatment, LV_ SFFV-VEGF165-T2A-tCD34 (VEGF165 cDNA co-expressed with a truncated form of CD34) was used. Vector constructions were used at 1:100 concentration for 48 h (virus titer 2.34e+08 TU/mL). MABs were subsequently sorted as a GFP + fraction. All vectors are constructed and produced by the Leuven Viral Vector Core. Details are described in S1. Details on the isolation and process of mesangioblasts can be seen in the Supplementary S1. Cytokine secretion profiling by cytokine antibody array. Rat Cytokine Antibody Array-Membrane (34 targets/ab133992, abcam, USA) was used to assess the efficacy of transduction and to profile the secretion of MABs allo-VEGF compared to MABs allo . Experiment was carried out in accordance with manufacturer's instructions. Details are described in Supplementary S2.

Animals: birth injury model and treatments.
The animal experiments were approved by the Animal Ethics Committee of the KU Leuven (P271-2015) and performed according to international guidelines. Seventyfour female virgin 12-week-old (250-300 g) Sprague-Dawley rats were used. Rats were anesthetized by intraperitoneal injection of ketamine (70 mg/kg), xylazine (7.5 mg/kg) and buprenorphine (0.05 mg/kg) and SVD was simulated by PNC + VD, as described previously 4,23 . One hour after SVD, rats were randomly assigned to receive an intra-aortic injection of 0.5 × 10 6 MABs (total volume = 0.8 mL) as described previously 14  www.nature.com/scientificreports/ method. We have previously documented the time course of events and functional recover after SVD using this method 24 . Rats were anesthetized with urethane (Sigma-Aldrich; 1.2 g/kg subcutaneously). During cystometry, the EUS was imaged using the Vevo 2100 micro-ultrasound platform and MS400 (30 MHz) probe (VisualSonics Inc., Toronto, Canada). Bursting activity of the urethral outlet reflects rhythmic opening and closing of the outlet to produce a pulsatile flow of urine 24 . Bursting of the EUS during micturition, which was confirmed by leakage of urine from the meatus, and coinciding with an increase in bladder pressure, was recorded in the motion mode (M-mode). A minimum of three micturition cycles were recorded per animal. Subsequent analysis was performed off-line with the Vevo LAB software (version 3.1.1). The frequency of EUS bursting was calculated and averaged over three cycles for each rat.
Vaginal strip contractility. Smooth muscle-strip contractility was determined using a standardized protocol 4 on the middle segment of the vagina, since that contains more smooth muscle tissue than the other segments. The contractile responses were normalized to tissue wet weight and to the maximum KCl response for the CA Figure 1. Timeline and design of the experiments: In the MABs auto group, a muscle biopsy is taken at 3-weeks of age, cells are isolated, expanded and re-injected in the same rat 9 weeks later. Control animals are injected with the same volume of vehicle (saline). In the MABs allo group, a muscle biopsy is taken from the gastrocnemius of a 3-weeks old rat and after isolation and expansion, the cell line is injected in a different rat than the donor, who is 12-weeks old. In the MABs allo-VEGF group, the same is done as in the MABs allo group, but cells are transduced with a lentiviral vector encoding VEGF. MABs auto autologous mesoangioblasts, MABs allo allogeneic mesoangioblasts, MABs allo-VEGF VEGF-overexpressed allogeneic mesoangioblasts. Control group: saline injection. *Primary outcome measure. Bioluminescence in vivo imaging. Cells were tracked in vivo by bioluminescence imaging (BLI) (IVIS Spectrum, Perkin Elmer) and images analyzed with software provided by the manufacturer (Living Image version 4.5). Rats were first anesthetized with 1.5% of isoflurane in 100% of oxygen and then given a single injection containing d-luciferin potassium salt dissolved in PBS (126 mg/kg, SC). Next, consecutive frames were acquired for five minutes until the maximum signal intensity was reached. A region of interest was drawn around the pelvic organs. The total flux (p/s) was measured within this region. BLI data was obtained 1d, 3d and 7d following MABs eGFP/fLUC injection.
Histology and immunohistochemistry. Whole pelvic floor organs were fixed in formalin, embedded in paraffin, cut into 5 µm sections, and stained with Masson trichrome. Alpha smooth muscle actin (α-SMA) staining was used to assess smooth muscle, Fast Myosin, a marker of type II muscle fibers, CD-34, a marker for endothelial cells, to assess vasculature and GAP-43, an axonal sprouting marker, used to evaluate peripheral nerve regeneration. All morphological analyses were performed using ImageJ software.

Results
MABs allo-VEGF boost functional urethral and vaginal recovery as compared to MABs allo . All rats injected with MABs allo functionally recovered by 14d, whereas only half of the controls recovered. Injection with MABs auto did not further improve that recovery ( Fig. 2A). MABs allo-VEGF enhanced the secretion of VEGF (5×) compared to untransduced MABs allo as measured by a rat cytokine array ( Supplementary Fig. 1). MABs allo-VEGF accelerated urethral function recovery in 33% of the rats at 7d. Only rats injected with MABs allo-VEGF showed a significant improvement of urethra and vaginal functional recovery at 14d ( Fig. 2A,B). Bioluminescence imaging (BLI) was used to determine the longitudinal fate of injected cells (Fig. 2C,D). Up to 7d, there was a progressive decay in BLI signal intensity. Animals injected with MABs allo-VEGF displayed a higher photon flux from the pelvis at 1 and 3d compared with MABs allo . Due to absence of significantly improvement using MABs auto , autologous cells were omitted from further experiments.
MABs promote muscle regeneration and neovascularization and MABs allo-VEGF additionally boost axonal sprouting. Morphometric analysis of the EUS was done at 7d (GAP-43), 14d and 60d (Fast Myosin, α-SMA and CD34). Both cell-treated groups showed a significantly higher expression of Fast Myosin and α-SMA, compared to saline injected rats at 14d (Fig. 3A,D,E). Only MABs allo-VEGF increased expression of these markers even further at 60d. Both cell-treated groups also showed increased neovascularization in urethral and vaginal specimens (CD34) at 14d and 60d (Fig. 3B,F, Supplementary Fig. 2A,B). They also showed enhanced axonal sprouting in the vagina at 7d (Supplementary Fig. 2C,D), however, in the urethra this effect was observed only in the MABs allo-VEGF injected rats (Fig. 3C,G).

MABs allo and MABs allo-VEGF contribute to urethral regeneration early on.
To gain a mechanistic insight into how MABs allo and MABs allo-VEGF treatment improves recovery, we analyzed the entire transcriptome from urethra tissue collected 3d and 7d after treatment using mRNAseq. Principal component analysis (PCA) and hierarchical clustering showed that urethra transcriptomes of MABs allo and MABs allo-VEGF -3d treated rats clustered, while saline 3d samples clustered with samples collected at 7d (Fig. 4A,B) 1)), respectively, of which 1741 were overlapping (Fig. 4C). A heat map showed that the majority of DEGs were upregulated at 3d in MABs treated rats and were downregulated at 7d and in controls (Fig. 4D).

MABs promote neuromuscular regeneration and modulate inflammatory response 3d after SVD.
Top Gene ontology (GO)-terms that were enriched in MABs allo upregulated genes were associated with myogenesis and dendrite development, while the pro-inflammatory process was associated with downregulated genes (Fig. 5A) (Fig. 4D). Interestingly, GO-terms enriched in both MABs allo and MABs allo-VEGF were associated with Rho-associated GTPases and epigenetic modifiers. Important up-and downregulated genes in both MABs allo and MABs allo-VEGF are displayed in Fig. 5B.

Comment
Herein we measured the neuromuscular regenerative effect of intra-arterial administration of MABs allo and MABs allo-VEGF in rats following SVD. Although rats show spontaneous functional urethral recovery by 21d 4,5 , there is a persistent loss of the periurethral and vaginal microvasculature 4 and dysfunction in the neuromuscular junction of the EUS 9 . Our most important findings were that injection of MABs induced earlier recovery of the EUS. Full functional recovery was paralleled by improved microvascularization and muscle regeneration in the urethra and vagina. Injection of MABs allo-VEGF fastened recovery (33% of animals at 7d), which was paralleled by enhanced axonal growth and improved the quality of recovery seen by the significant improve of urethral and vaginal function at 14d. Interestingly, MABs allo-VEGF also showed a longer-term effect seen by significantly higher muscle content at 60d compared to controls. This is the first study reporting the functional effect of MABs in the rat model for SVD. Similarly to other studies using stem cells 8,25-28 , we observed urethral function recovery and improvement in neovascularization and muscle regeneration at 14d after intra-arterial MABs injection. This improvement coincides with evidence of wound healing activity early on (3d) on mRNAseq such as upregulation of Rho/GTPase activity, epigenetic www.nature.com/scientificreports/ factors, muscle and dendrite development. MABs have a regenerative potential for muscular disorders due to their capacity to differentiate into skeletal and smooth muscle. The low survival and poor engraftment of stem cells is one of the obstacles for effective tissue regeneration 29,30 . We hypothesized that autologous MABs would persist longer or engraft more, hence improve functional recovery. Herein we did not observe that, which is in line with earlier experiments 31,32 . Low engraftment has been previously explained by the unfavorable ischemic microenvironment leading to acute apoptosis 33 as well as loss of cell-adhesion potential inherent to cell preparation 29 .
The improved recovery of the urethra and the vagina, even with the transient presence of the MABs, suggests that the beneficial effect may be mediated primarily through paracrine effects rather than the engraftment. If so, such an effect may also be achieved by administration of drugs, which activate pathways studied herein such as the Rho/GTPase, epigenetic modifiers or others that mimic the observed downstream effect.
One important soluble growth factor secreted by MABs is VEGF, certainly when dealing with ischemic injuries. Therefore, we assessed the additional effect of VEGF, which sped up EUS recovery, and apparently improved nerve regeneration. This may be because of increased cell survival observed at 1d and 3d post injection since VEGF is known for promoting cell survival in a hypoxic environment 34 . VEGF may also be neuroprotective in ischemic injury, e.g. because of its angiogenic effect or its direct trophic effects on neuronal survival 19 . On the molecular level, urethras of MABs allo-VEGF rats showed up regulation of genes that control neuronal development and small GTPases pathways at 3d. Small GTPases are master regulators of actin cytoskeleton rearrangements, which is important for axonal sprouting 35 . On the other hand, Sox2, Il1b and Nrg1, genes involved in dedifferentiation of Schwann cells 36,37 , were among the few genes that were upregulated only in MABs allo-VEGF group at 3d. It is tempting to assume that the activation of these pathways/genes most probably played a role in faster nerve sprouting of the urethra observed at 7d that resulted to a thicker skeletal and smooth muscle fibers observed at 60d. Similarly, other studies using peripheral nerve injury models demonstrated increased muscle volume in animals efficiently re-innervated 38,39 .
Given the clinical feasibility of using allogeneic MABs allo taken from a biobank and immediate availability, our study aimed to investigate the potential merits of allogeneic cell therapy as a more pragmatic approach in patients exhibiting indications at delivery, such as prolonged second stage of labor, high birth weight, or evident trauma confirmed by ultrasound examination. Although autologous cell therapy is ideal and remains a viable strategy, particularly in patients with early indications of being at high risk for stress urinary incontinence (e.g. prior urinary incontinence), it would require all patients to donate cells in advance. Therefore, exploring the use of allogeneic cells from a biobank offers a more practical alternative, as these cells can be readily obtained when needed without relying on individual patient donations.
One limitation of our study is that we did not provide evidence for a paracrine effect. In future experiments this will be important, because our strategy employs gene-modified cells. Though gene modified MSCs are considered to be safe, amenable for repeated treatments and effective, the strategy is still not applied in clinic. Nevertheless, this is the first study reporting functional effects of MABs in a solid rat model for vaginal birth injury, as well as the added effects of delivering VEGF to the site of the injury. Moreover, we used a robust urethral function as outcome measure, shown earlier to be reliable and operator-independent 24 .
We concluded that Intra-arterial injection of MABs allo-VEGF enhances neuromuscular regeneration induced by untransduced MABs allo and accelerates the functional urethral and vaginal recovery after SVD.